The present invention relates to novel combinations of proteins and to their use as medicament. More particularly, it relates to the preparation of vaccines against CMV.
Human cytomegalovirus (CMV), an enveloped virus with a 230 kbp DNA double strand, is the largest virus of the herpesvirus family. Like the other members of this virus family, it exists in latent form and can undergo repeated reactivation steps which lead to a viremia several years after the initial infection. CMV is widely distributed throughout the world and, while being well tolerated by healthy individuals, it is associated with pathologies which frequently have drastic consequences for the fetus and for immuno-depressed patients (transplant, AIDS and cancer patients) (see Review (1)).
Following a primary infection during pregnancy, the vertical transmission of the virus to the fetus via the placenta leads to complications in the newborn. These are, in particular, sensorial disorders (vision, hearing) and significant mental backwardness which arise during the first few years of the child""s life. Infection with CMV is associated with graft rejection in transplant patients (foreign transplants of marrow, kidneys, heart, liver). It constitutes one of the most drastic opportunistic infections in HIV+ patients which, despite antiviral chemotherapy, fall victim toxe2x80x94frequently lethalxe2x80x94pathologies. All these reasons mean that CMV infection raises a substantial public health problem.
The use of unspecific antivirals, such as Ganciclovir* and Foscarnet* during transplantation causes cytotoxicity problems. The secondary effects frequently require a reduced dosage or termination of the treatment, thus posing the risk of the disorder associated with the viral infection recurring. In certain cases, the intravenous injection of high doses of immunoglobulins has reduced the frequency of pneumopathies and rejections. Attempts at adoptive immunotherapy have been developed by injecting, into recipients, marrow of clones of cytotoxic T-cells which are specific for CMV (2).
The development of a subunit vaccine would be of enormous importance to future mothers. In fact, it has been demonstrated that maternal immunity which has been acquired prior to conception could protect the newborn babies from the damage of congenital infection (3). The development of an anti-CMV immunity in transplant patients would be an important factor against the development of infection-associated diseases. Vaccination trials with the attenuated virus of the Towne strain were carried out and are being performed on seronegative volunteers and seronegative transplant patients who have received a transplant from seropositive donors (4). In the majority of the cases, the vaccination has reduced the severity of the diseases associated with viral replication. As the pathogenic activity of such vaccines has not definitively been excluded and where the use of live vaccines might cause severe side effects in immunodeficient individuals, novel approaches using recombinant viral proteins in the presence of adjuvants are being developed.
The importance for public health of developing a vaccine against CMV no longer has to be demonstrated as a result of the problem presented by the congenital infection, which has hitherto been underestimated by the medical community.
The savings made by developing a strategy of preventing diseases linked to CMV infection in risk patients would be substantial. In fact, estimates of the costs associated with the vaccination of an individual and its taking (serological analyses, vaccine, treatment of minor side effects) show that it would be approximately 50 times less than that of the care with which a newborn is provided which is the victim of congenital infection. CMV infection can be observed in ⅔ kidney transplant patients and more often in other transplant patients. Taking into consideration that the infection is associated with complications in approximately ⅓ of these, the annual costs in addition to the transplantation costs are considerable. Despite the impact of drugs such as Ganciclovir*, the injection of gammaglobulins or the transfer of anti-CMV T-clones on the disease, prevention of the primary infection and reactivation in these patients should be a priority. The benefits of an immunization are obvious at the clinical and at the economic level.
The applicant has now found that the combination of CMV protein pp65 or a fragment thereof with another hapten or antigen of CMV allows the immune reaction towards this virus to be potentiated, in particular by stimulating the compartments T CD4+ and CD8+ against the virus.
This is why the present invention relates to a fusion protein, characterized in that it comprises at least part of the cytomegalovirus (or CMV) protein pp65, or of a protein having at least 80% homology with protein pp65, in combination with at least one second peptide fragment derived from CMV.
Protein pp65 is a CMV matrix protein which is internalized in the cells and delivered into the cytosol at the same time as the virion, very shortly after infection.
It can be used whole or in the form of one or more fragments; the peptide fragments which make up the chimeric protein preferably have a length of greater than, or equal to, 9 amino acids and cover different HLA class I restrictions. The applicant has shown that a peptide of protein pp65 of a length greater than 9 amino acids can be internalized by a presenting cell and presented to a CD8+ specific T-line by a class I MHC molecule. The importance of constructions which include these peptides and the protein IE1 for vaccination would be linked to the use of antigens which are capable of entering the presenting cell by a unique endocytosis pathway, simultaneously inducing it to a TCD4+ and TCD8+ response.
The second peptide fragment which is present in the fusion protein is preferably composed of protein IE1 or one of its epitopes, or of a protein having at least 80% homology.
In fact, the polypeptide sequence of IE1, which is a major regulatory protein of the viral cycle, is highly conserved between the different viral strains. Introduction of this protein into a subunit vaccine would allow the induction of memory CD4+ helper T-cells, which are capable of cooperating with the induction of cytotoxic CD8+ T-cells against pp65 and with the production of antibody against the envelope protein gB, subjected to greater variability (cross help). In fact, it has been shown that most of the neutralizing antibodies which are present in the serum of infected individuals were directed against the viral envelope glycoprotein gB (UL55). This is why protein gB was considered to be one of the most important viral antigens for vaccination. A large number of protocols in which recombinant viruses are used (adenovirus, vaccinia, canarypox) have been developed (5, 6, 7).
The problems presented by these vaccines are linked either to the pathogenic character of the live viruses or to the fact that they induce too low antibody titers. An alternative is the use of recombinant antigens which are combined with adjuvants. The combination of glycoprotein gB with the chimeric protein IE1-pp65 into one structure allows its conformation to be maintained, which is indispensable for its immunogenicity, would offer a means for stimulating the T and B compartments of the antiviral response. Moreover, activation of the CD4+ T-cells against IE1, which is mediated by the IE1 peptides in combination with class II MHC molecules being recognized, would allow viral replication in the adjacent cells, which are subjected to the effect of TNFxcex3 and TNFxcex1 (8) to be controlled. Naturally, these hypotheses are not intended to limit the scope of the invention.
In particular, use can be made of fragment e4 of CMV protein IE1, or a peptide fragment having at least 80% homology with said fragment e4. e4, or exon 4, which comprises 406 amino acids, is a fragment of protein IE1, which is composed of 491 amino acids.
According to another advantageous aspect of the invention, the fusion protein comprises
a) the fragment delimited by the amino acid residues 162 and 175 of the sequence of protein IE1, or
b) a peptide fragment having at least 90% homology with said fragment mentioned under a).
Other epitopes are also suitable for carrying out the invention, such as those mentioned by Davignon et al. (8).
The fusion protein according to the invention can additionally contain a peptide fragment derived from a microorganism other than CMV and/or any polypeptide fragment which allows it to be purified later from Tag-type sequences; these sequences, which are placed upstream or downstream of the protein of interest allow it to be purified or labeled; the use of xcex2-galactosidase, histidine hexamers (His6) or GST may be mentioned as examples. According to a preferred embodiment, the fusion protein comprises a peptide fragment derived from an enzyme with glutathion-S transferase (or GST) activity.
The GST protein will make it easier, in particular, to purify the fusion protein from a complex culture medium.
The invention therefore relates to a chimeric protein GST-IE1-pp65 of 145 kd, which can be prepared in E. coli. Its immunogenicity was demonstrated in vitro by the proliferation of IE1-specific CD4+ T-cell-clones and by the lysis of target cells incubated in the presence of a pp65-specific CD8+ T-line. The applicant has shown that the protein in soluble form and its fragments allow the CD4+ and CD8+ T-compartments of the specific cell response to be stimulated in vitro. These results make this protein the reactant of choice for designing a subunit vaccine.
The nucleotide sequences which code for a fusion protein as defined hereinabove are also within the scope of the invention.
The fusion proteins and the corresponding nucleotide sequences can be used as medicament and in particular for the preparation of a vaccine for preventing infections caused by CMV. Such a vaccine will be suitable for inducing an efficient response against a primary infection before the virus has replicated actively.
In accordance with another aspect, the invention relates to a pharmaceutical composition, characterized in that it contains
a) at least part of the CMV protein pp65 or of a protein having at least 80% homology with protein pp65, in combination with at least one second peptide fragment derived from CMV, or
b) nucleotide sequences coding for the peptides mentioned under a).
In addition to formulation excipients known to the skilled worker, such as stabilizers, preservatives, antioxidants, adapted to suit the route of administration, in particular an injection, the compositions can contain immunity adjuvants. They can also contain other CMV epitopes. Finally, they can be formulated with systems which improve transport and presentation of the molecules to the target cells.
The proteins can be in the form of various different proteins in the composition; they can also be in the form of a fusion protein as described above; the same applies to the corresponding nucleotide sequences.
In addition, the composition can contain other epitopes, in particular CMV envelope antigens, such as protein gB.
In accordance with yet another aspect, the invention relates to a process for the preparation of a fusion protein, characterized in that the following steps are carried out:
a) a first DNA sequence which codes for at least a part of the CMV protein pp65 is linked with a second DNA sequence which codes for another polypeptide or protein derived from CMV so as to obtain a recombinant DNA sequence which codes for a fusion protein,
b) the recombinant DNA sequence is introduced into a construction containing the elements required for its expression, and, if appropriate, sequences which code for other polypeptides,
c) the construction obtained in b) is introduced into host cells which are subsequently cultured under conditions in which the expression system of the fused DNA is functional, so that the fusion protein is produced in the host cell,
d) the fusion protein produced in the host cell is recovered and purified.
The host cell which contains a nucleotide sequence coding for a fusion protein, which can be obtained in the process described hereinabove, also comes within the scope of the invention. This host cell can be selected in particular from the group consisting of the bacteria, the viruses, the yeasts and eukaryotic cells, in particular higher eukaryotes.
Other characteristics and advantages of the invention will become obvious from the examples which follow.